This invention relates to methods and kits useful for determining the length of nucleic acid repeat sequences. More specifically, this invention relates to methods and kits useful for determining the length of nucleic acid repeat sequences by employing a discontinuous primer extension reaction.
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Methods for the analysis of genetic polymorphism have found wide utility in basic research, clinical diagnostics, forensics, and other areas. One particularly useful method of detecting genetic polymorphism is based on variations in the length of repeat sequences, such methods being variously referred to as short tandem repeat analysis (STR), variable number of tandem repeat analysis (VNTR), minisattelite analysis, and microsatellite analysis.
Detection of length polymorphisms in nucleic acid repeat sequences has up to now relied on gel electrophoresis for the determination of the length of the repeat sequence. However, gel electrophoresis has several important drawbacks in the context of repeat sequence length polymorphism analysis. First, molecular length measurements based on electrophoretic mobility are inherently imprecise due to a complicated relationship between molecular size and electrophoretic mobility. Second, the degree to which the electrophoretic process can be multiplexed is limited by the number of electrophoresis lanes and by the size of different loci run in a single lane, i.e., loci must be selected which do not electrophoretically co-migrate.
The method of the present invention comprises a discontinuous primer extension reaction wherein a primer is extended in discrete increments such that in each increment of primer extension the primer is extended by an amount corresponding to a single repeat unit. Following each increment of discrete primer extension, a detection step is performed in which a modulation in a signal is detected when the primer has been extended by an amount equal to the total length of a repeat region. Thus, by counting the number of increments of discrete primer extension required to cause a modulation in the signal, the number of repeat units making up the repeat region is determined.
It is an object of the present invention to provide a precise and reproducible method for determining the number of repeat units making up a repeat region of a nucleic acid repeat sequence.
It is another object of the present invention to provide a method for determining the number of repeat units making up a repeat region of a nucleic acid repeat sequence which can perform a large number of measurements in parallel.
It is yet an additional object of the present invention to provide a method for determining the number of repeat units making up a repeat region of a nucleic acid repeat sequence which does not require an electrophoretic separation.
It is an object of the present invention to provide kits and reagents useful for practicing a method for determining the number of repeat units making up a repeat region of a nucleic acid repeat sequence having the above described characteristics.
In a first aspect, the foregoing and other objects of the invention are achieved by a method for determining the number of repeat units in a repeat region of a target nucleic acid comprising annealing a primer-complementary portion of a target nucleic acid to a primer thereby forming a target-primer hybrid; performing a first primer extension reaction using a first primer extension reagent; separating the target-primer hybrid and unreacted first primer extension reagent; performing a second primer extension reaction using a second primer extension reagent, wherein at least one of the first or second primer extension reagents includes an extendible nucleotide having a label attached thereto; separating the target-primer hybrid from unreacted second primer extension reagent; measuring a signal produced by the label; treating the label so as to render the label undetectable; and repeating the above steps until the signal is substantially less than a signal detected in a previous cycle.
In one preferred embodiment of the first aspect of the invention, the step of performing a second primer extension reaction farther includes reacting the target-primer hybrid with a primer termination reagent.
In yet another preferred embodiment of the first aspect of the invention, the label is a fluorescent or chemiluminescent molecule.
In another preferred embodiment of the first aspect of the invention, the label is attached to the extendible nucleotide through a cleavable linker.
In an additional preferred embodiment of the first aspect of the invention, the target nucleic acid is amplified prior to analysis. Preferably such amplification is achieved using a PCR.
In an another preferred embodiment of the first aspect of the invention, the step of treating the label so as to render the label undetectable includes either cleaving the label from the labeled extendible nucleotide or destroying a signal producing property of the label.
In another preferred embodiment of the first aspect of the invention, the target-primer hybrid is attached to a solid support. Preferably, one of the primer or the target nucleic acid is attached to the solid support.
In a novel, preferred embodiment employing a solid support, the invention includes a method for determining the number of repeat units in a repeat region of a target nucleic acid comprising the steps of:
(A) contacting a plurality of different-sequence primers with a polynucleotide sample under conditions effective for the primers to anneal to primer-complementary regions in one or more target polynucleotides, to form one or more target-primer hybrid(s), wherein either (1) each different-sequence primer contains (i) a target binding segment and (ii) a tag segment having a nucleotide sequence that uniquely identifies the target binding segment, or (2) one or more polynucleotides in the sample are tagged polynucleotides that contain a tag segment having a nucleotide sequence that uniquely identifies the attached polynucleotide,
(B) performing a first primer extension reaction on the hybrid(s) using a first primer extension reagent;
(C) separating the target-primer hybrid(s) and unreacted first primer extension reagent;
(D) performing a second primer extension reaction on the hybrid(s) using a second primer extension reagent, wherein at least one of the first or second primer extension reagents includes an extendible nucleotide having a label attached thereto;
(E) separating the target-primer hybrid(s) from unreacted second primer extension reagent;
(F) measuring a signal produced by the label;
(G) treating the label so as to render the label undetectable; and
(H) repeating a cycle of steps (A) through (G) until the signal detected in the target-primer hybrid(s) is substantially less than a signal detected in a previous cycle,
wherein (I) prior to step (F), at least an aliquot of either (1) the different-sequence primers or (2) the tagged sample polynucleotides are contacted with an addressable array of immobilized, different tag complements, and each different tag complement contains a sequence that is complementary to one of the tag segments, under conditions effective to hybridize the tag segments to corresponding tag complements on the support.
In one embodiment, the contacting in step (I) is performed prior to step (A). In another embodiment, the contacting in step (I) is performed after step (A), and/or before any one of steps (B), (C), (D), (E), and (F). In yet another embodiment, steps (A) through (H) are performed on at least two replicate arrays, and one of the replicate arrays is subjected to at least one more cycle of steps (A) through (G) than is a second replicate array.
In a second aspect, the foregoing and other objects of the invention are achieved by a method for determining the number of repeat units in a repeat region of a target nucleic acid comprising annealing a primer-complementary portion of a target nucleic acid to a primer thereby forming a target-primer hybrid; performing a first primer extension reaction using a first primer-extension reagent; separating the target-primer hybrid from unreacted first primer extension reagent; performing a second primer extension reaction using a second primer extension reagent and with a primer termination reagent, the primer termination reagent including a nucleotide terminator having a label attached thereto; separating the target-primer hybrid from unreacted second primer extension reagent and unreacted primer termination reagent; measuring a signal produced by the label; and repeating the above steps until a signal is detected indicating incorporation of the labeled nucleotide terminator into the primer extension product.
In yet another preferred embodiment of the second aspect of the invention, the label is selected from the group consisting of fluorescent and chemiluminescent molecules.
In an additional preferred embodiment of the second aspect of the invention, the target nucleic acid is amplified prior to analysis. Preferably such amplification is achieved using a PCR.
In a preferred embodiment of the second aspect of the invention, the target-primer hybrid is attached to a solid support. Preferably, one of the primer or the target nucleic acid is attached to the solid support.
In a novel, preferred embodiment, the invention includes a method for determining the number of repeat units in a repeat region of a target nucleic acid comprising the steps of:
(A) contacting a plurality of different-sequence primers with a polynucleotide sample under conditions effective for the primers to anneal to primer-complementary regions in one or more target polynucleotides, to form one or more target-primer hybrid(s), wherein either (1) each different-sequence primer contains (i) a target binding segment and (ii) a tag segment having a nucleotide sequence that uniquely identifies the target binding segment, or (2) one or more polynucleotides in the sample are tagged polynucleotides that contain a tag segment having a nucleotide sequence that uniquely identifies the attached polynucleotide,
(B) performing a first primer extension reaction on the hybrid(s) using a first primer-extension reagent;
(C) separating the target-primer hybrid(s) from unreacted first primer extension reagent;
(D) performing a second primer extension reaction on the hybrid(s) using a second primer extension reagent and with a primer termination reagent, the primer termination reagent including a nucleotide terminator having a label attached thereto;
(E) separating the target-primer hybrid(s) from unreacted second primer extension reagent and unreacted primer termination reagent;
(F) measuring a signal produced by the label; and
(G) repeating a cycle of steps (A) through (F) until a signal is detected indicating incorporation of the nucleotide terminator,
wherein (H) prior to step (F), at least an aliquot of either (1) the different-sequence primers or (2) the tagged sample polynucleotides, are contacted with an addressable array of immobilized, different tag complements, and each different tag complement contains a sequence that is complementary to one of the tag segments, under conditions effective to hybridize the tag segments to corresponding tag complements on the support.
In one embodiment, the contacting in step (H) is performed prior to step (A). In another embodiment, the contacting in step (H) is performed after step (A), and/or before any one of steps (B), (C), (D), (E), and (F). In yet another embodiment, steps (A) through (G) are performed on at least two replicate arrays, and one of the replicate arrays is subjected to at least one more cycle of steps (A) through (F) than is a second replicate array.
In a third aspect, the foregoing and other objects of the invention are achieved by a kit useful for determining the number of repeat units in a repeat region of a target nucleic acid comprising a primer having a sequence complementary to a primer-complementary portion of a target nucleic acid; a first primer extension reagent; and a second primer extension reagent, wherein at least one of the first or second primer extension reagents includes an extendible nucleotide having a label attached thereto.
In a preferred embodiment of the third aspect of the invention, the primer is attached to a solid support.
In an additional preferred embodiment of the second aspect of the invention, the label is selected from the group consisting of fluorescent and chemiluminescent molecules.
In another preferred embodiment of the second aspect of the invention, the label is attached to the extendible nucleotide through a cleavable linker.
These and other objects, features, and advantages of the present invention will become better understood with reference to the following description, drawings, and appended claims.